Shaken cultures are usually batch-cultures where all the components are added at the beginning of the cultivation. Too high substrate concentration, inadequate aeration, uncontrolled growth, synthesis of harmful metabolites (over-flow or anaerobic metabolisms), catabolite repression, and even substrate intoxication easily emerge in non-controlled shaken cultures. The biomass yield in shaken E. coli cultures is typically in the range of 1-2 g/l (dry weight), in microscale often much lower. With well-designed substrate feeding and pH control up to 50-fold higher biomass can be produced in commonly used conventional laboratories or industrial bioreactors. Control strategies (continuous monitoring and controlling) as applied in larger scale are not easily applicable in small shaken cultures. Setup of continuous monitoring and feeding is difficult to realize in the small scale. Non-controlled growth and insufficient aeration will fast bring oxygen-depletion. During oxygen limitation fermentation products (acetate, CO2, formic acid, lactic acid, ethanol, succinic acid) are formed in quantities which inhibit the growth of bacteria and impair recombinant protein processes. Some of these metabolites can be synthesized also under aerobic conditions, if glucose uptake and glycolysis are faster than the capacity of the citric acid cycle. During such over-flow metabolism acetyl-CoA is transformed into acetate which is secreted to the culture medium. Catabolic repression of respiration (Crabtree effect) can occur also during long-term exposure to high glucose concentrations even in aerobic conditions.
To avoid oxygen limitation, over-flow metabolism and the Crabtree effect, high-cell-density cultivations in bioreactors normally apply the fed-batch technology. In substrate-limited fed-batch cultivation the bacterial growth rate can be controlled with one limiting substrate (typically glucose which is used as the sole carbon source). Oxygen consumption increases relative to respiratory activity (substrate usage) and growth rate. Therefore, by suppressing growth of microbes by substrate-limitation also oxygen limitation can be avoided. However the majority of simple cultivations are done in shake flasks without monitoring or feeding possibilities. The applied cultivation method is usually batch-cultivation. The biomass yield typically remains low, and the quality of the so-produced culture is non-predictable and often poor. High-cell-densities are not achieved with the batch method because such cell densities would require so high initial consentration of nutrients that it would be toxic to the microbe.
Since measuring and feeding devices are not normally applicable for simple shaken cultures, alternative strategies have been developed. In medical therapies drugs are often supplied by slow release of a substrate over a long period of time. Drug delivery-like systems are rarely applied in microbial cultivations, as the substrates normally are small molecules such as glucose and ammonia, for which the release rate is difficult to control. With a “drug-delivery” like system Lübbe et al. (Appl Microbiol Biotechnol (1985) 22: 424-427) have supplied NH4Cl in Streptomyces clavuligerus cultivation and recently Jeude et al. (Biotechnol Bioeng (2006) Vol. 96, No. 3:433-443) have used silicone elastomer (polydimethylsiloxane) disks containing glucose to create fed-batch like conditions for cultivations (see also Büchs et al. WO 2006/119867 “Fermentation method and apparatus for its implementation”) These disks can be added to cultivation vessels, but they are not integrated parts of them. However, relatively small amounts of glucose can be packed into such a matrix. Furthermore, the glucose release rate from such matrix is usually fastest in the beginning of cultivation, when the amount of microorganisms is lowest and the risk for over-flow metabolism is highest. This may explain, why such systems have not rapidly become popular in simple cultivations of the biotechnologically most important bacterial species, Escherichia coli. 
Tyrell et al. (J. Bacteriol 75 (1958): 1-4; “Biphasic system for growing bacteria in concentrated culture”) have presented a method to pack nutrients like yeast extract into a gel. This method however has no possibilities of controlling the release rate of nutrients, especially glucose-release from such a gel occurs extremely fast. Therefore it is not applicable for high-cell-density cultivation. This method never became popular, since nutrient-rich well-buffered complex cultivation media like super broth or terrific broth are easier to use and provide higher cell densities.
An interesting application for animal cell cultures has been presented by Green and James (U.S. Pat. No. 3,926,723 “Method of controllably releasing glucose to a cell culture medium” 1975). They used small amount of soluble starch as the carbon source for cells. The horse, pig or bovine serum used in cultivation medium provided enough catalytic activity to release gradually small amounts of glucose. Also added enzymes could be used instead of the serum enzymes. This approach used only 2 g/l of starch, which in theory would support max 1 g/l of biomass (cells). In human cell cultures no significant increase of cell number was obtained. It was not used for controlling or limiting culture growth rate, but instead it was only used to prevent accumulation of one growth-retarding compound, lactic acid. Thus the authors seem not to be aware of the fed-batch technology as a strategy for reaching high cell densities and with the low concentration of starch they used their method can not be regarded as a high-cell-density cultivation. For bacterial cultivations (e.g. for Escherichia coli) this approach will not work: complex cultivation media such as serum, yeast extract or peptones contain several components which can function as a carbon-source. Therefore growth control can not be obtained by limiting the concentration of one carbon source (e.g. glucose). In microbial fed-batch cultivations, chemically defined media and only one growth-limiting substrate are typically used at a time. Furthermore, as will be shown in accordance of the present invention, only small amounts of starch can remain soluble in the medium to provide enough glucose for high-cell-density microbial cultivations. For aerobically growing microbes the presence of high amounts of starch severely weakens the oxygen transfer capacity of the medium and thereby increases the risks of anaerobic metabolism. For this reason, an intelligent system to pack high amount of carbon source into culture vessels is required.
The slow-release approaches so far published are limited in 1) scalability, 2) the amount of the delivered substrate that can be packed to the system or 3) methods to accurately control the substrate-release. None of the above-described methods provide an integrated solution for all of these essential requirements for high-cell-density cultivation of microbes in simple shaken cultures. In methods based on enzymatic degradation of a substrate delivering polymer, high amounts of polymer must be loaded to the cultivation vessel without impairing the properties (e.g. oxygen transfer capacity) of the cultivation medium.